Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

Abstract

Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium tuberculosis isolates were collected retrospectively from Shenzhen Center for Chronic Disease Control in 2009 and 127 isolates from project on anti-tuberculosis drug resistance surveillance in Shenzhen during 2007 to 2009.Drug susceptibility to rifampin and isoniazid of the stains were determined by DNA microarray, and results were compared to that obtained with reference proportion method drug susceptibility testing for sensitivity, specificity and accuracy.The consistency of microarray and phenotypic susceptibility testing was evaluated by Kappa test.Genetic mutations in rpoB, katG, inhA，regulatory region of inhA, and regulatory region of ahpC were investigated by DNA sequencing to assess proper loci for rapid molecular diagnosis. Results Compared against results of proportion method, the sensitivity, specificity and accuracy of the DNA microarray assay for rifampin resistance were 94.4%, 97.5% and 96.0% respectively, and for isoniazid resistance were 79.1%, 100% and 86.8% respectively.Mutations in resistance-determining region of rpoB were observed in 97.2%（70/72） of the isolates resistant to rifampin, which contributed in the 531, 526, 516, 511 and 533 codon region.Mutations in katG315 codon, inhA-15, and ahpC regulatory region were found in 70.3%（64/91）, 11.0%（10/91） and 9.9%（9/90） of the isolates resistant to isoniazid, respectively.Mutations of ahpC promoter region consists of ahpC-9 (4 strains), ahpC-10 (2 strains), ahpC-6 (2 strains), ahpC-12 (1 strain), and ahpC-32 (1 strain). Conclusions DNA microarray provided a rapid method for the detection of drug-resistant Mycobacterium tuberculosis isolates, and demonstrated good performance except less sensitive for isoniazid resistance.The mutations in ahpC regulatory region might be good target loci for detection of isoniazid-resistant Mycobacterium tuberculosis, so screening the region may significantly improve the sensitivity for molecular genetic tests.（Chin J Lab Med,2012,35:1125-1129） Key words: Mycobacterium tuberculosis; Drug resistance; bacterial; Rifampin; Isoniazid; Oligonucleotide array sequence analysis